Successful transcriptional silencing of the majority of each eukaryotic genome is critical because inappropriate gene expression leads to disastrous consequences, including oncogenic transformation. The long-term goal of the proposed research is to understand fundamental mechanisms of transcriptional silencing. Discovery of homologs of the yeast silencing gene SIR2 from organisms as diverse as prokaryotes and mammals established an important new focus for studying silencing. Recent progress provides the first evidence for SIR-like silencing in humans and suggests that SIR2 silencing functions may be separated genetically. The primary goals of the research proposed are designed to address long-standing unanswered questions about SIR2 itself and specific questions and hypotheses raised in initial characterization of functionally conserved silencing domains. The project aims are to: .Extend biochemical and cell biological analyses of the SIR2 silencing protein. Pursue analysis of Sir2 protein domains identified for their significance in silencing and its specificity. Identify S1R2 interacting molecules in order to understand the mechanism of Sir2p's activity and regulation of its function. These aims will be accomplished through genetic, biochemical and cell biological approaches. Results obtained will lead to greater knowledge of silenced chromatin complexes and mechanisms of transcriptional control and genomic regulation conserved from yeast to humans. Progress made understanding these centrally important regulatory mechanisms may ultimately lead to improved diagnostics and refined therapeutic and genetic interventions for human diseases resulting from loss of gene regulation.